(1) Field of the Invention
The present invention relates to transformed dairy cultures with a natural 7.8-kb plasmid pSRQ700 which was isolated from Lactococcus lactis subsp. cremoris DCH-4, a known strain. pSRQ700 encodes a restriction/modification system named LlaII. When introduced into a phage-sensitive dairy culture, such as L. lactis, pSRQ700 confers strong phage resistance against the three most common lactococcal phage species: 936, c2 and P335 found in dairy product fermentations. The LlaII endonuclease was purified and found to cleave the palindromic sequence 5'/GATC-3'. The low copy plasmid pSRQ700 was mapped and the genetic organization of LlaII localized. Cloning and sequencing of the entire LlaII system allowed the identification of three open reading frames. The three genes (LlaIIA, LlaIIB, and LlaIIC) overlapped and are under one promoter. A terminator was found at the end of LlaIIC. The genes LlaIIA and LlaIIB coded for m.sup.6 A-methyltransferases and LlaIIC for an endonuclease. The native LlaII R/M system from Lactococcus lactis is also expressed by and conferred strong phage resistance to various industrial S. thermophilus strains. Resistance was observed against phages isolated from yogurt and Mozzarella wheys. This is the first demonstration of increased phage resistance in S. thermophilus.
(2) Description of Related Art
Lactococcus lactis and Streptococcus salivarius subsp. thermophilus cultures are used extensively worldwide in the manufacture of fermented dairy products. The cultures are normally inoculated into pasteurized or heat-treated milk to quickly start and control the fermentation. In this non-sterile milk environment, the added cells come into contact with the wild bacteriophage population that has survived pasteurization. Although natural phage concentration is low, their population increases very rapidly if phage-sensitive cells are present in the starter culture. The consequent lysis of a large number of sensitive cells retards the fermentation process. To cope with this natural phenomenon, the dairy industry has developed a series of solutions including the use of phage resistant Lactococcus lactis strains (Hill, C., FEMS Microbiol. Rev. 12:87-108 (1993)).
Lactococcus lactis
In the last decade, extensive research was conducted on interactions between lactococcal phage and their hosts. Lactococcus lactis was found to possess many plasmids coding for natural defense mechanisms against bacteriophages. Over 40 plasmids with phage defense barriers have been identified. Phage resistance systems are classified into three groups based on their mode of action: blocking of phage adsorption, restriction/modification and abortive infection. Phage-resistant Lactococcus lactis strains have been constructed by introducing these natural plasmids into phage-sensitive strains (Sanders, M. E., et al., Appl. Environ. Microbiol. 40:500-506 (1980)). The conjugative abilities of some of these plasmids was exploited to construct such resistant strains (Harrington, A., et al., Appl. Environ. Microbiol. 57:3405-3409 (1991); Jarvis, A. W., et al., Appl. Environ. Microbiol. 55:1537-1543 (1988); Sanders, M. E., et al., Appl. Environ. Microbiol. 52:1001-1007 (1986); and Ward, A. C., et al., J. Dairy Sci. 75:683-691 (1992)). However, after considerable industrial use of these strains, new phages capable of overcoming the introduced defense mechanism have emerged (Alatossava, T., et al., Appl. Environ. Microbiol. 57:1346-1353 (1991); Hill, C., et al., J. Bacteriol. 173:4363-4370 (1991); and Moineau, S., et al., Appl. Environ. Microbiol. 59:197-202 (1993)). Thus, the search for different natural phage barriers is still an ongoing objective for dairy product starter culture manufacturers.
Over the years several studies have established the heterologous nature of the lactococcal phage population (Jarvis, A. W., et al., Intervirology 32:2-9 (1991)). Based on electron microscopy and DNA hybridization studies, the Lactococcal and Streptococcal Phage Study Group, which is part of the International Committee on Taxonomy of Viruses, reported the existence of 12 different lactococcal phage species. Recently, this number has been reduced to 10 due to the reclassification of the 1483 and T187 species into the P335 species. Strong DNA homology is observed among members of the same species but no homology is found between species (Braun, V., et al., J. Gen. Microbiol. 135:2551-2560 (1989); Jarvis, A. W., et al., Intervirology, 32:2-9 (1991); Moineau, S., et al., Can. J. Microbiol. 38:875-882 (1992); Powell, I. A., et al., Can. J. Microbiol. 35:860-866 (1989); and Prevots, F., et al., Appl. Environ. Microbiol. 56:2180-2185 (1990)). Although many species have been isolated, only three appear to be the most problem for the dairy industry. The species 936 (small isometric head) and c2 (prolate head) have been, by far, the most disturbing lactococcal phage species worldwide. Interestingly, phages from the P335 species (small isometric head) are now being isolated with increasing frequency from North American dairy plants (Moineau, S., et al., Appl. Environ. Microbiol. 59:197-202 (1993)). Two recent surveys revealed that 100% of the 45 lactococcal phages isolated from Canadian cheese plants and U.S. buttermilk plants were classified within one of these three species: 22 phages belonged to the 936 species, 18 to the c2 species and 5 to the P335 species (Moineau, S., et al., J. Dairy Sci. 77:18 suppl. 1 (1994); and Moineau, S., et al., Can. J. Microbiol. 38:875-882 (1992)). Therefore from a practical point of view, industrial Lactococcus lactis strains should at least be resistant to the three most common phage species: 936, c2 and P335. Due to the diversity of lactococcal phages, the need for phage defense mechanisms with broad activity (attacking many species) is becoming more meaningful. Because of the characteristics of phages, restriction/modification (R/M) systems have the potential to fulfill this objective.
The phenomenon of R/M was first reported more than 40 years ago (Luria, S. E., et al., J. Bacteriol. 64:557-569 (1952)) and received a molecular explanation ten (10) years later (Bickle, T. A., et al., Microbiol. Rev. 57:434-450 (1993); and Dussoix, D., et al., J. Mol. Biol. 5:37-49 (1962)). The main biological activity of R/M is believed to be in preventing the entrance of foreign DNA (including phage DNA) into the cell. These gatekeepers are roughly the prokaryotic equivalent of the immune system (Wilson, G. G., Nucleic Acids Res. 19:2539-2566 (1991)). There are currently more than 2400 known restriction enzymes and over 100 have been cloned and sequenced (Raschke, E., GATA 10:49-60 (1993); and Roberts, R. J., et al., Nucleic Acid Res. 21:3125-3137 (1993)). There are several kinds of R/M systems and they appear to have equivalent biological activities but achieved in different ways. At least four types of R/M systems have been identified: I, II, IIs, and IIII (Bickle, T. A., et al., Microbiol. Rev. 57:434-450 (1993); Wilson, G. G., Nucleic Acids Res. 19:2539-2566 (1991); and Wilson, G. G., et al., Annu. Rev. Genet. 25:585-627 (1991)). Of these, type II is the simplest and the most common. Illustrative patents are European Patent Application 0 316 677, European Patent Application 0 452 224, U.S. Pat. Nos. 4,530,904 to Hershberger, et al, 4,883,756 to Klaenhammer et al, 4,931,396 to Klaenhammer et al and 5,019,506 to Daly et al.
Many R/M systems have been characterized at the protein level. Restriction enzymes are very dissimilar, suggesting an independent evolution and not from a common ancestor (Bickle, T. A., et al., Microbiol. Rev. 57:434-450 (1993); Wilson, G. G., Nucleic Acids Res. 19:2539-2566 (1991); and Wilson, G. G., et al., Annu. Rev. Genet. 25:585-627 (1991)). In contrast, extensive similarities occur among the methyltransferases (Bickle, T. A., et al., Microbiol. Rev. 57:434-450 (1993); Klimasauskas, S., et al., Nucleic Acids Res. 17:9823-9832 (1989); Lauster, R., J. Mol. Biol. 206:313-321 (1989); McClelland, M., et al., Nucleic Acids Res. 20:2145-2157 (1992); Wilson, G. G., Nucleic Acids Res. 19:2539-2566 (1991); and Wilson, G. G., et al., Annu. Rev. Genet. 25:585-627 (1991)). They can be grouped into three classes corresponding to the modification types: m.sup.4 C, m.sup.5 C and m.sup.6 A (Wilson, G. G., Nucleic Acids Res. 19:2539-2566 (1991); and Wilson, G. G., et al., Annu. Rev. Genet. 25:585-627 (1991)). m.sup.4 C and m.sup.6 A can be further divided in two (.alpha. and .beta.) and three (.alpha., .beta., and .gamma.) subclasses respectively, based on their amino acid sequences (Klimasauskas, S., et al., Nucleic Acids Res. 17:9823-9832 (1989); and Lauster, R., J. Mol. Biol. 206:313-321 (1989)).
A number of plasmids encoding for R/M have been identified in Lactococcus (Hill, C., FEMS Microbiol. Rev. 12:87-108 (1993)). Surprisingly, only a handful have been partially characterized. The LlaI R/M system encoded on the conjugative plasmid pTR2030, isolated from Lactococcus lactis subsp. lactis ME2, was the first analyzed at the sequence level (Hill, C., et al., J. Bacteriol. 173:4363-4370 (1991)). The methylase gene of pTR2030 system has been sequenced and the deduced protein was found to share similarities with the type-IIs methyltransferase (m.sup.6 A), M. FokI (Hill, C. L., et al., J. Bacteriol. 173:4363-4370 (1991)). The endonuclease genes have also been sequenced and four open reading frames were identified (O'Sullivan, D. J., et al., FEMS Microbiol. Rev. 12:P100 (1993)). Recent data have provided evidence for a new class of multisubunit endonucleases (O'Sullivan, D. J., et al., FEMS Microbiol. Rev. 12:P100 (1993)). The restriction complex, however, has yet to be purified and its recognition sequence is unknown.
ScrFI was the first classical type II restriction enzyme isolated from Lactococcus lactis and is the only one commercially available (Fitzgerald, G. F., et al., Nucleic Acid Research. 10:8171-8179 (1982)). ScrFI recognizes the sequence 5'-CCNGG-3' where N is any of the nucleotide. Two methylase genes from the Lactococcus lactis subsp. lactis UC503 chromosome have been cloned and sequenced (Davis, R., et al., Appl. Environ. Microbiol. 59:777-785 (1993); and Twomey, D. P., et al., Gene 136:205-209 (1993)). They both coded for a m.sup.5 C MTase. The endonuclease gene has yet to be identified. Mayo et al (Mayo, B., et al., FEMS Microbiol. Lett. 79:195-198 (1991) isolated a type II endonuclease (also named LlaI) from L. lactis subsp. lactis NCDO497 which recognized the sequence 5'-CCWGG-3 (W is A or T) but the R/M genes have not been cloned.
Recently Nyengaard, N., et al, Gene 136, 371-372 (1993) described LlaI and LlaBI, which are type II restriction endonucleases from Lactococcus lactis subsp. cremoris W9 and W56. These endonucleases recognize DNA sequences 5'/GATC-3 and 5'-C/TRYAG3', respectively. The plasmids from these strains were transformed into a plasmid free and endonuclease negative Lactococcus lactis subsp. lactis by electroporation to produce a transformed strain which resisted phage attack. The DNA was not isolated and sequenced and the natural plasmid was used for the transformation. Further, the authors did not indicate if the plasmids encoded methyl transferase. Strains W9 and W56 were not tested.
Streptococcus thermophilus
Similar information on phage and phage resistance is still very limited for Streptococcus thermophilus despite sustained phage infections in the yogurt and Mozzarella cheese industry (Mercenier et al, Genetic engineering of lactobacilli, leuconostocs and Streptococcus thermophilus, In M. J. Gasson and W. M. DeVos (ed.), Genetics and biotechnology of lactic acid bacteria. Blackie Acad. Prof. Glaskow, UK p. 253-293 (1994)). Fortunately, S. thermophilus phages are much more closely related to each other than the L. lactis phages. It appears that there is only one S. thermophilus phage species (Mercenier et al Genetic engineering of lactobacilli, leuconostocs and Streptococcus thermophilus, In M. J. Gasson and W. M. DeVos (ed.), Genetics and biotechnology of lactic acid bacteria. Blackie Acad. Prof. Glaskow, UK p. 253-293 (1994)). Only very few phage defense mechanisms have been reported for S. thermophilus. Four chromosomally-encoded type II R/M systems have been identified in S. thermophilus. Solaiman and Somkuti (Solaiman, D. K. Y., et al., FEMS Microbiol. Lett. 67:261-266 (1990); and Solaiman, D. K. Y., et al., FEMS Microbiol. Lett. 80:75-80 (1991)) have isolated the endonuclease Sth134I and Sth117I which are isoschizomers of HpaII and EcoRII, respectively. Benbadis et al (Benbadis, L., et al., Appl. Environ. Microbiol. 57:3677-3678 (1991)) and Guimont et al (Guimont, C., et al., Appl. Microbiol. Biotechnol. 39:216-220 (1993)) have isolated the endonucleases sslI and Sth455I, respectively. Both are also isoschizomers of EcoRII. In addition, S. thermophilus might possess abortive-like phage defense mechanisms (Larbi et al. J. Dairy Res. 59:349-357 (1992)), although definitive proof has yet to be demonstrated. None of the R/M systems so far identified in S. thermophilus have been cloned, sequenced, or used in commercial strains for improvement of phage resistance. There is believed to be no report on improvement of phage resistance of S. thermophilus strains.